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PubMed1996

The flare in serum alkaline phosphatase activity after orchiectomy: a valuable negative prognostic index for progression-free survival in prostatic carcinoma.

PURPOSE: We determined whether an early flare in serum alkaline phosphatase activity after orchiectomy was of prognostic value for progression-free survival in patients with advanced prostatic carcinoma. MATERIALS AND METHODS: A retrospective analysis of a data base from a Dutch multicenter study on prostatic carcinoma was done to determine the prognostic value of a flare in alkaline phosphatase activity after orchiectomy in 112 patients with metastatic (75%) or locally advanced (25%) disease. Cox's proportional hazards models and Kaplan-Meier survival curves were used. RESULTS: Of the patients 50% had initially increased alkaline phosphatase levels and a flare in activity was demonstrated in 87% 2 to 4 weeks after orchiectomy. The prostate specific antigen nadir (cutoff 4 ng./ml.) 6 months after orchiectomy was of significant prognostic value for progression-free survival. A flare in alkaline phosphatase activity after orchiectomy demonstrated an early significant prognostic value for progression-free survival, independent of the serum alkaline phosphatase activity. CONCLUSIONS: The simplicity, ready availability and cost-effectiveness of serum alkaline phosphatase activity as a prognostic index render it attractive to the clinician, particularly early in the course of prostatic carcinoma. Measuring the flare in alkaline phosphatase activity within 1 month of orchiectomy may permit early identification of patients in whom the disease is likely to progress rapidly and who would potentially benefit from aggressive treatment.

Aged↗
PubMed1979

Acid phosphatase: new developments.

Acid phosphatase was the first "tumor marker" to be measured in the blood, and over 40 years have passed since an elevation of the serum acid phosphatase level was observed in patients with prostatic carcinoma. However, significant elevations in the level of this enzyme have been observed in other diseases, as well as elevations of other tissue phosphatases. Many improvements in the colorimetric technique have been introduced, but none has been used successfully to detect the tissue origin of this ubiquitous enzyme. The finding that prostatic acid phosphatase is antigenically distinct from acid phosphatase of other tissues opened a new horizon in the measurement of acid phosphatase in prostatic cancer. On the basis of this immunochemical specificity, several immunoassays have been employed for determining the prostatic acid phosphatase level.

Acid Phosphatase↗
PubMed1976

Histochemical and biochemical study on acid phosphatase activity in differentiating liver of chick.

1. Acid phosphatase activity at different ontogenetic stages of liver in chick has been studied both by histochemical and quantitative biochemical methods. 2. It has been observed that the activity of acid phosphatase does not remain constant, but undergoes changes at different phases of epigenesis. The activity rhythmically becomes higher and lower throughout the whole period of epigenesis. Comparatively higher activities have been found up to 8 days of incubation, and then the activities become lower up to 14 days and again increased at later periods of development. 3. Higher acid phosphatase activity has been observed in the perivascular connective tissue than the parenchymal cells from the 8th to the 20th day of incubation. 4. The acid phosphatase activity is high in the nuclei of early developing red blood corpuscles, but in advanced stage of R.B.C. the phosphatase activity is very high in the cytoplasm, and it is practically absent in the nuclei. 5. The activity of acid phosphatase may have some relation with the cell death and catabolic processes during chick liver morphogenesis. The relation of the phosphatase activity with the growth and differentiation of the liver has been discussed.

Acid Phosphatase↗
PubMed2001

Pentapeptide regulation of aspartyl-phosphate phosphatases.

Aspartyl-phosphate phosphatases are integral components of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The Rap and Spo0E families of protein phosphatases specifically dephosphorylate the sporulation response regulators Spo0F and Spo0A, respectively. The phosphatases interpret regulatory signals antithetical to sporulation and the Rap phosphatases are subject to inactivation by specific pentapeptides generated from an inactive peptide precursor. Additional regulatory signals are brought about by the complex activation circuit that generates the Phr pentapeptide inhibitors of Rap phosphatases. Phr peptide's recognition of the Rap phosphatase targets is remarkably specific. Specificity is dictated by the amino acid sequence of the pentapeptide. The identification of tetratricopeptide repeats in the Rap proteins may explain the mechanism by which Phr peptides bind to and inhibit the activity of Rap phosphatases.

Amino Acid Sequence↗
PubMed2000

Genetic and biochemical characterization of a protein phosphatase with dual substrate specificity in Streptomyces coelicolor A3(2).

A gene encoding a protein phosphatase (SppA) with a phosphoesterase motif, which was predicted by the genome project of the Gram-positive bacterium Streptomyces coelicolor A3(2), was cloned by PCR in pET32a(+) and expressed in Escherichia coli. SppA fused to thioredoxin (TRX-SppA) showed distinct heat-stable phosphatase activity toward p-nitrophenyl phosphate with optimal pH 8.0 and optimal temperature 55 degrees C. Mn2+ greatly enhanced enzyme activity, as is found with other protein Ser/Thr phosphatases. TRX-SppA was not inhibited by sodium orthovanadate or okadaic acid, both of which are known to be specific inhibitors of protein phosphatases. TRX-SppA showed phosphatase activity toward not only phosphoThr (pThr) and pTyr but also oligopeptides containing pSer, pThr, and pTyr, indicating that SppA is a protein phosphatase with dual substrate specificity. Disruption of the chromosomal sppA gene resulted in severe impairment of vegetative growth. All of these observations show that SppA, a protein phosphatase with dual specificity, plays an important, but not essential, role in vegetative growth of S. coelicolor A3(2). The presence of a single copy of sppA in all the 13 Streptomyces species examined, as determined by Southern hybridization, suggests a common role of SppA in general in Streptomyces species.

Amino Acid Sequence↗
PubMed1990

A study of the acid phosphatase of Treponema denticola.

This study describes some of the properties of the acid phosphatase of the potential periodontopathogen Treponema denticola. The highest enzyme activity was found in 87 h old cells. Two optimum pHs for enzyme activity were detected, one at pH 4.8 and another at pH 6.2. Divalent cations did not influence the acid phosphatase of T. denticola. The anion F- added in the form of NaF and at a level greater than 20 micrograms/ml F- diminished the activity of the acid phosphatase of intact cells of T. denticola. The addition of 10 micrograms/ml F- as SnF2 induced a statistically significant reduction of acid phosphatase activity. The apparent Km for the acid phosphatase was 7.3 mM with p-nitrophenyl phosphate as substrate. Fluoride appeared to be a noncompetitive inhibitor of the enzyme with an apparent Ki of 0.3 mM. Acid phosphatase may be released partially in osmotic shock fluids. Also, 7-diazonium-1, 3-naphthalene disulfonate, which is incapable of penetrating the bacterial permeability barrier and is known to inactivate enzymes found in the bacterial periplasmic place, suppressed the activity of the acid phosphatase in intact cells of T. denticola.

Acid Phosphatase↗
PubMed1996

Biochemical properties and excretion behavior of repressible acid phosphatases with altered subunit composition.

Yeast repressible acid phosphatase (rAP) is the oligomeric extracellular enzyme encoded by the three structural genes PH05 (p60), PHO10 (p58) and PHO11 (p56). We examined the ability of acid phosphatases formed by various subunit combinations to be excreted into the medium. Plasmids with repressible acid phosphatase structural genes under control of the yeast glyceraldehyde-phosphate dehydrogenase (GAP) promoter were constructed to obtain constitutive expression of acid phosphatase, and yeast strains with disruptions in PHO5, PHO10 and PHO11, respectively, were used to generate mutants expressing single genes or specific gene combinations. EndoF treatment of acid phosphatases, produced by these strains, followed by SDS-electrophoresis in combination with densitometry techniques revealed that the ratio p60/(p56 + p58) among structural polypeptides in extracellular enzyme is constant and equals to 6.0. A study of acid phosphatases formed by single type subunits was undertaken. Expression products of PHO5, PHO10 and PHO11 genes were isolated from the culture medium. The specific activities of the enzymes were found to be 33, 2 and 2 mM x mg-1 x min-1, respectively. The values of Mr estimated by HPLC chromatography for the enzymes encoded for by the genes PHO5, PHO10 and PHO11 and SDS-polyacrilamide gel electrophoresis data suggested an oligomeric organisation of the enzymes. Isoelectric focusing in polyacrylamide gel with immobilised pH gradient followed by activity staining yielded numerous sharp bands of homopolymeric acid phosphatases forms being different in their pI. The kinetic characterisation of the enzymes revealed differences in Km values, sensitivity to temperature inactivation, inhibition by orthophosphate and the effect of pH on the enzyme activity.

Acid Phosphatase↗
PubMed1998

Characterization of an exocellular protein phosphatase with dual substrate specificity from the yeast Yarrowia lipolytica.

In previous work, the major endocellular protein phosphatase activity has been identified in the secretory yeast Yarrowia lipolytica as a PP2A. The aim of the present work was to seek the presence of one protein phosphatase excreted in the exocellular medium and to study its activity during yeast growth in media supplemented or not supplemented with inorganic phosphate. Protein phosphatase was purified and activity was assayed by following the dephosphorylation of three substrates, [32P]casein, phosphotyrosine and a synthetic tyrosine-phosphorylated peptide. Phosphatase activity recovered in the medium after 25 h culture was greatly enhanced by Pi-deficiency. After several purification steps, the enzyme preparation presents an apparent electrophoretic homogeneity on SDS-PAGE with associated phosphoseryl/threonyl and phosphotyrosyl activities. The kinetic properties exclude contamination by a copurified protein and it is concluded that the two activities are carried by the same single proteic species. It was characterized by gel filtration as a 33 kDa protein with one single subunit demonstrated by SDS-PAGE. An absolute requirement for reducing-agents is observed suggesting that the enzyme contains at least one essential reactive cysteinyl residue. Optimum pH value is 6.1, apparent K(m) for phosphotyrosine was calculated to be 760 microM and Hill coefficient 3.2 indicating a rather high cooperativity. These results showed that the involvement of alkaline and/or acid phosphatase was unlikely. In conclusion, a protein phosphatase distinct from endocellular PP2A is secreted by Yarrowia lipolytica and characterized as a phosphotyrosine protein phosphatase with associated phosphoseryl/threonyl activity.

Amino Acid Sequence↗
PubMed2002

Novel caged fluorescein diphosphates as photoactivatable substrates for protein tyrosine phosphatases.

We have characterized some novel caged fluorescein diphosphates as photoactivatable, cell-permeable substrates for protein tyrosine phosphatases and explored their usefulness in identifying inhibitors of tyrosine phosphatases. 1-(2-Nitrophenyl)ethyl protected fluorescein diphosphate (NPE-FDP) undergoes rapid photolysis to release FDP upon irradiation with a 450-W UV immersion lamp and its by-product does not inactivate protein tyrosine phosphatase 1B (PTP1B) or alters the viability of cells. The generated FDP from photolysis of NPE-FDP was shown to have exactly the same properties as FDP, which can be used as a PTP substrate in pure enzyme assays. We have also demonstrated that the PTP activity can be measured using NPE-FDP in small droplets. Its advantage as an inert substrate before photolysis allows the possibility of applying nanospray technology in screening and optimizing PTP inhibitors through a large chemical library. Like other caged bioeffectors such as nucleotide and inositol trisphosphate, NPE-FDP is cell-permeable. The NPE-FDP can be photolyzed to generate FDP inside cells, and then can be hydrolyzed by phosphatases to produce fluorescein monophosphate and subsequently to fluorescein. Although Jurkat cells contain high concentrations of CD45, it has not been possible to use FDP as a substrate to measure CD45 activity in the intact cell. This is due to the hydrolysis of FDP by several other cellular phosphatases. However, NPE-FDP can be useful as a cell-permeable substrate for overexpressed phosphatases such as alkaline phosphatase.

Animals↗
PubMed1995

Characterization of the recombinant C-terminal domain of dystrophin: phosphorylation by calmodulin-dependent protein kinase II and dephosphorylation by type 2B protein phosphatase.

We report that the C-terminal domain of skeletal muscle dystrophin expressed as a fusion protein with glutathione S-transferase (designated GST-CT-1) is a substrate for Ca2+/calmodulin-dependent phosphorylation and dephosphorylation. GST-CT-1 and GST-CT-1F (GST-CT-1 truncated by 20-25 residues) were phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). The stoichiometries of phosphorylation by CaM kinase II were 1.65 mol of Pi/mol of GST-CT-1 and 0.39 mol of Pi/mol of GST-CT-1F, respectively, suggesting that the principal site(s) of phosphorylation is (are) located in the C-terminal 20-25 residues that are missing from GST-CT-1F. The GST-CT-1 fusion protein was phosphorylated on both serine and threonine residues, whereas GST-CT-1F was phosphorylated only on serine. CaM kinase II-phosphorylated GST-CT-1 and GST-CT-1F were efficiently dephosphorylated by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (type 2B protein phosphatase). Importantly, calcineurin was found to be associated with a purified sarcolemmal membrane preparation enriched in dystrophin. Type 2A protein phosphatase isolated from smooth muscle (SMP-I) and its catalytic subunit (SMP-ic) also dephosphorylated GST-CT-1, but were less active toward these substrates than was calcineurin. Type 2C phosphatase (SMP-II) and type 1 protein phosphatases [SMP-III, SMP-IV, and myosin-associated phosphatase (PP1M) of smooth muscle and skeletal muscle protein phosphatase 1c] were ineffective in dephosphorylating the C-terminal region of dystrophin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Binding of a protein-tyrosine phosphatase to DNA through its carboxy-terminal noncatalytic domain.

The noncatalytic domain of a non-receptor-type protein-tyrosine phosphatase (the T-cell phosphatase or PTP-S) isolated from a rat spleen cDNA library shows homology with the basic domains of transcription factors Fos and Jun [Swarup, G., Kamatkar, S., Radha, V., & Rema, V. (1991) FEBS Lett. 280,65-69]. We have expressed this phosphatase in Escherichia coli under the control of T7 promoter. The PTP-S gene product expressed in E. coli shows protein-tyrosine phosphatase activity and binds to DNA at pH 7.4 as determined by DNA affinity chromatography, Southwestern blotting, and gel retardation methods. The carboxy-terminal region of this phosphatase was fused with glutathione S-transferase by constructing expression vectors. Experiments using fusion proteins with glutathione S-transferase suggest that the carboxy-terminal 57 amino acids of PTP-S are sufficient for DNA binding. Deletion of the C-terminal 57 amino acids of PTP-S protein abolished its DNA binding property, as determined by Southwestern blotting, but not its enzymatic activity. This suggests that the C-terminal 57 amino acids are essential for the DNA binding function of this protein but not for its enzymatic activity. Another non-receptor-type protein-tyrosine phosphatase, PTP-1, when expressed in enzymatically active form in E. coli did not bind to DNA. These results suggest that a nontransmembrane protein-tyrosine phosphatase, PTP-S, binds to DNA in vitro through its carboxy-terminal noncatalytic region.

Amino Acid Sequence↗
PubMed1990

Receptor interconversion model of hormone action. 2. Requirement of both kinase and phosphatase activities for conferring estrogen binding activity to the estrogen receptor.

Three interconvertible forms of the estrogen receptor have been identified in the oviduct of estrogen-stimulated chicks. The non-estradiol binding form (Rnb) can be converted to the lower affinity binding form (Ry, Kd = 0.8 nM) by a process requiring the gamma-phosphoryl moiety of ATP. The enzymatic activity (Fy) essential for this "receptor potentiation" has been isolated from oviduct cytosol using ammonium sulfate fractionation, DEAE chromatography, and HPLC size-exclusion chromatography. The potentiation appears to require both kinase and phosphatase activities. The Fy kinase characteristically phosphorylates casein, histones, and glycogen synthase. Comparison of the kinase with casein kinase II, which also phosphorylates casein and glycogen synthase, indicates that Fy represents a distinct protein kinase since its activity is not stimulated by spermine or inhibited by heparin. Fy-mediated conversion of Rnb to Ry is blocked by the phosphatase inhibitors vanadate, fluoride, and pyrophosphate. The substrate specificity of the Fy phosphatase activity is distinct from that of the two well-characterized protein phosphatases 1 and 2A. Moreover, the requirement for Fy phosphatase activity in converting Rnb to Ry could not be mimicked by its substitution with purified protein phosphatases 1 or 2A. The unique substrate specificity of the oviduct protein phosphatase and protein kinase, which are apparently necessary to confer estradiol binding characteristics to the receptor, implies that these enzymes play a key role in the control of the estrogen receptor in its function as a transcription factor.

Amino Acids↗
PubMed1980

Role of phosphatidylinositol in attachment of alkaline phosphatase to membranes.

The mechanism of release of alkaline phosphatase from membranes by phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was studied. Alkaline phosphatase was readily released from pig kidney microsomes by phospholipase C but not by a variety of other treatments, e.g., high ionic strength, extremes of pH, divalent cations, chelating agents, or analogues of the polar head group of phosphatidylinositol. Alkaline phosphatase released from microsomes by phospholipase C did not bind to phospholipid vesicles containing phosphatidylinositol. Alkaline phosphatase solubilized from microsomes by butanol extraction, however, was able to bind phospholipid vesicles even when they contained no phosphatidylinositol. The ability of butanol-extracted alkaline phosphatase to bind to phospholipid vesicles was destroyed by added phosphatidylinositol-specific phospholipase C. Hydrolysis of added phosphatidylinositol by endogenous phospholipase activity in butanol extracts was also accompanied by loss of binding ability. Loss of binding ability was paralleled by a decrease in the apparent molecular weight of alkaline phosphatase. These results indicate that alkaline phosphatase is attached to membranes by a strong interaction with phosphatidylinositol.

Alkaline Phosphatase↗
PubMed1996

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by phospholipids.

Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by phospholipids was examined using purified enzyme and Triton X-100/phospholipid-mixed micelles. Anionic phospholipids activated phosphatidate phosphatase activity whereas zwitterionic phospholipids had a slight inhibitory effect on activity. Cardiolipin (A0.5 = 1.9 mol %), CDP-diacylglycerol (A0.5 = 2.6 mol %), and phosphatidylinositol (A0.5 = 5.5 mol %) were the most potent anionic phospholipid activators. Enzyme activation by cardiolipin (n=2.8), CDP-diacylglycerol (n=2.1), and phosphatidylinositol (n=3.3) followed positive cooperative kinetics. A kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase activation by anionic phospholipids. The dependence of phosphatidate phosphatase on phosphatidate was cooperative (n approximately 2.2) in the absence and presence of phospholipid activators. Cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were mixed competitive activators of phosphatidate phosphatase activity. The major effect of the activators was to cause a decrease in the Km for phosphatidate. Sphinganine, a positively charged sphingoid base, inhibited phosphatidate phosphatase activity and antagonized the activation of the enzyme by cardiolipin and phosphatidylinositol. Sphinganine caused an increase in the cooperativity of cardiolipin activation, but had little effect on the A0.5 value for cardiolipin. On the other hand, sphinganine had little effect on the cooperativity of phosphatidylinositol activation, but caused an increase in the A0.5 value for phosphatidylinositol. The activation constants for cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were within the range of their cellular concentrations. These results suggested that the activation of phosphatidate phosphatase activity by anionic phospholipids may be physiologically relevant.

Cardiolipins↗
PubMed1979

Evidence that three structural genes code for human alkaline phosphatases.

The number of structural gene loci that code for the different molecular forms of human alkaline phosphatase is unknown. Physical properties of the enzymes, immunological data, chemical inhibition and genetic studies suggest that at least three structural genes are involved: one coding for alkaline phosphatase from placenta, another for the enzyme from intestine, and one or more for the enzymes from liver, kidney and bone. Badger and Sussman have shown that alkaline phosphatases from human liver and placenta are products of different structural genes, and Greene and Sussman have shown that alkaline phosphatase from a metastasised bronchogenic carcinoma was nearly identical to the enzyme from placenta. However, other tumour-associated alkaline phosphatases and the enzymes from normal tissue other than placenta and liver have not been identified by conclusive structural criteria, and thus it is not known whether these onco-alkaline phosphatases represent ectopic production or unusual post-translational modification of the enzymes found in normal tissues. We present here, using a sensitive peptide-mapping technique, structural evidence that the enzyme forms from liver, kidney and serum from a patient with Paget's disease of bone (osteitis deformans) are products of the same structural gene and can be easily distinguished from either the intestinal or placental isoenzymes. The technqiue seems to be useful for the classification of tumour-associated alkaline phosphatases on a structural basis.

Alkaline Phosphatase↗
PubMed1969

Heterogeneity of liver acid phosphatases in developing chick embryo.

1. The development, localization and heterogeneity of acid phosphatase and a Zn(2+)-activated acid phosphatase in cellular fractions of developing chick liver were studied. 2. Acid phosphatase is distributed abundantly in the particulate and soluble fractions. The soluble fraction is rich in Zn(2+)-activated acid phosphatase, which attains its peak activity at about 15 days of incubation. 3. The particulate acid phosphatase activity is inhibited by fluoride but not by sodium l(+)-tartrate or cysteine. On the other hand, the soluble Zn(2+)-activated acid phosphatase activity is inhibited by sodium l(+)-tartrate and cysteine but not by fluoride. 4. The pH optimum of these two enzymes is similar at about 5.6. 5. The soluble Zn(2+)-activated acid phosphatase activity appears to be thermally stabilized by the treatment with Triton X-100 or bovine serum albumin.

Acid Phosphatase↗
PubMed1988

Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes.

Desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes results in a 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of beta-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37 degrees C, pH 8.0. In both preparations alkaline phosphatase treatment significantly decreased desensitization of agonist-stimulated adenylate cyclase activity by 40-75% (P less than 0.05). Similar results were obtained after alkaline phosphatase treatment of membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized duck erythrocytes. Moreover, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with 12-O-tetradecanoylphorbol 13-acetate returned agonist-stimulated adenylate cyclase activity to near control values. In all experiments, inclusion of 20 mM-sodium phosphate to inhibit alkaline phosphatase during treatment of membranes attenuated the enzyme's effect on agonist-stimulated adenylate cyclase activity. In addition, alkaline phosphatase treatment of membranes from control and isoprenaline-desensitized turkey erythrocytes increased the mobility of beta-adrenergic-receptor proteins, specifically photoaffinity-labelled with [125I]iodocyanopindolol-diazirine, on SDS/polyacrylamide-gel electrophoresis. The increased mobility of the beta-adrenergic-receptor proteins after alkaline phosphatase treatment of membranes was again inhibited by 20 mM-phosphate. These results provide additional evidence for a direct role for phosphorylation in desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes.

Adenylyl Cyclases↗
PubMed1996

Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia.

In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA cannot phosphorylate P63, whereas either PKG or the casein kinase phosphorylate P63 in vitro. On the basis of these findings we propose that a protein phosphatase/kinase system is involved in the regulation of exocytosis in P. tetraurelia cells.

Animals↗